The vertebrate inner is the most complex of the sense organs responsible for hearing and balance. Yet during development it forms from a simple epithelium, the otic placode. During development of the ear, multipotent progenitor cells become progressively restricted in their potential. This process is controlled by regulatory genes whose temporal and spatial expression patterns are tightly regulated in an orchestrated gene regulatory network (GRN). We and others have established a hierarchy of events controlling the specification and determination of inner ear progenitors and identified some of the regulatory genes involved. Based on this information, we have established a preliminary network controlling these processes and designed a molecular screen to identify novel otic specifiers. We have defined co-regulated groups of genes that reflect different stages of ear specification. We will now harness genome sequence information, new technology to monitor changes in the expression of more than 100 genes simultaneously and newly developed bioinformatics tools to build up and verify the preliminary GRN. Specifically we will: identify new genes responsive to otic inducing signals establish the epistatic relationships between ear specific transcription factors and signaling components isolated and characterize enhancers controlling transcription factor expression in the ear and examine direct inputs predict common upstream regulators for genes in each synexpression group using newly developed algorithms and test the predicted regulators in vivo. This will uncover the basic GRN controlling the specification of inner ear progenitors together with its terminal target genes. In the future, this GRN will serve as a basis for studying protein-protein and protein-DNA interactions to build up a complete network, for quantitative analysis and mathematical modeling of this process, as well as a platform to discover new candidate genes for human disease affecting hearing and balance. PUBLIC HEALTH RELEVANCE: The sense of hearing is crucial for communication with our environment. In newborn babies and young children it is essential for normal development, development of social interactions and in particular for the acquisition of speech. Worldwide 1.64 children per 1000 births are born deaf or with some form of hearing impairment mainly due to inner ear defects causing sensorineuronal deafness. Congenital deafness is acquired through drugs or infection during pregnancy in 25% of affected children. In recent years, much progress has been made in identifying the etiology of other cases, in particular through large scale mutagenesis screens in mice, leading to the successful identification of many deafness genes: known genetic mutations now account for about 50% of hearing defects. However, the underlying causes of the remaining 25% are still unknown. Thus, there is clearly a need to identify novel candidate genes and to analyze their function during ear formation and their relationship to specific phenotypes. This project aims to do so by uncovering the molecular events that gradually commit multipotent, naive cells to inner ear fate. We have harnessed our understanding of the biology of the process and newly available tools for large-scale expression analysis to design a molecular screen and have now defined new factors that control inner ear specification. These are good candidates for congenital deafness. We now plan to build up a network of genes involved in this process by using newly developed bioinformatics tools combined with in vivo functional analysis. In the long term, this will lead to the development of better diagnostic tools, novel strategies for prevention of deafness and for genetic counseling and be important for stem cell research connected to age-related hearing loss.